Treatment and detection of inherited neuropathies and associated disorders

ABSTRACT

The present disclosure relates to methods of detecting and treating inherited neuropathy.

CROSS REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCEOF MATERIAL SUBMITTED ELECTRONICALLY

This application is a Continuation of International Patent ApplicationNo. PCT/US2020/031708, filed May 6, 2020, which claims the benefit ofU.S. Provisional Patent Application No. 62/844,370, filed May 7, 2019,and U.S. Provisional Patent Application No. 62/987,151, filed Mar. 9,2020, each of the foregoing are incorporated herein by reference intheir entireties.

GRANT FUNDING DISCLOSURE

This invention was made with government support under grant numbersNS065712, NS075764 and GM119018 awarded by the National Institutes ofHealth (NIH). The government has certain rights in the invention.

SEQUENCE LISTING

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: Filename: 761350_000281_SL.txt; Size: 147,687bytes; Created: Dec. 15, 2021.

FIELD OF THE INVENTION

The present disclosure relates to methods of detecting and treatinginherited neuropathy.

BACKGROUND

Peripheral neuropathies are amongst the most frequent neurodegenerativediseases, with diabetic neuropathy and hereditary origins amongst themost common mechanisms of action. For the inherited neuropathies, alsoknown as Charcot-Marie-Tooth disease (CMT), the remaining diagnostic gapof patients is ˜50%. In our understanding, CMT represents an umbrellaconcept for clinically and genetically heterogeneous inherited monogenichighly phenotypically penetrant conditions affecting the peripheralnerves. CMT is classified depending on conduction velocity asdemyelinating (CMT1) and axonal (CMT2) types. Distal hereditary motorneuropathy (dHMN) represents a form of CMT2 in which the burden ofdisease falls predominantly or exclusively on motor nerves (Rossor,Tomaselli, and Reilly 2016). A similar condition includes ALS4 (juveniledHMN+brisk reflexes as sign of upper motoneuron involvement). As opposedto CMT1, for which over 90% of cases have mutations in known genes, only20 to 30% of CMT2 and distal HMN patients receive a genetic diagnosis(Fridman et al. 2015).

SUMMARY

The disclosure provides a method of treating and/or detecting inheritedneuropathy. In various aspects, the method comprises detecting thepresence of a mutation in the sorbitol dehydrogenase (SORD) gene in asample from a subject. In various embodiments, the SORD mutation is aDNA variant classified as pathogenic or likely pathogenic according toAmerican College of Medical Genetics and Genomics (ACMG) criteria.Optionally, the method comprises diagnosing the subject with inheritedneuropathy when the presence of a mutation in the SORD gene is detected.Optionally, the method comprises administering to the subject acomposition that comprises an agent selected from the group consistingof an aldose reductase inhibitor; an aldose reductase antisenseoligonucleotide; a polynucleotide that encodes a SORD peptide; a SORDpeptide; an agent that blocks expression of a mutant SORD gene; and anagent that corrects the mutation in SORD gene. In various aspects, themethod comprises administering to the subject Alrestatin, Epalrestat,Diepalrestat, Fidarestat, Imirestat, Lidorestat, Minalrestat,Ponalrestat, Ranirestat, Salfredin B₁₁, Sorbinil, Tolrestat, Zenarestat,or Zopolrestat (or a combination thereof). In various aspects, themethod comprises administering to the subject an aldose reductaseantisense oligonucleotide; a polynucleotide that encodes a SORD peptide;an agent that blocks expression of a mutant SORD gene; an agent thatcorrects the mutation in SORD gene; or a combination of any of theforegoing. In various aspects, the method comprises administering to thesubject a SORD peptide. Administration of a combination of any of theforegoing is also contemplated. Optionally, the method comprisesmeasuring sorbitol levels in a sample from the subject.

Also provided is use of an (i) aldose reductase inhibitor (e.g.,Alrestatin, Epalrestat, Diepalrestat, Fidarestat, Imirestat, Lidorestat,Minalrestat, Ponalrestat, Ranirestat, Salfredin B₁₁, Sorbinil,Tolrestat, Zenarestat, and/or Zopolrestat); (ii) an aldose reductaseantisense oligonucleotide, a polynucleotide that encodes a SORD peptide,an agent that blocks expression of a mutant SORD gene, and/or an agentthat corrects the mutation in a SORD gene; and/or (iii) a SORD peptidefor the treatment of inherited neuropathy (or use in the preparation ofa medicament for treatment of inherited neuropathy) in a subject whichhas been tested for the presence of a mutation in the sorbitoldehydrogenase (SORD) gene.

The disclosure further provides a method of characterizing a neuropathyin a mammalian subject, the method comprising measuring the level ofsorbitol in a subject suffering from a neuropathy, wherein a sorbitollevel of greater than about 10 g/L indicates that the neuropathy isassociated with a mutation in the sorbitol dehydrogenase (SORD) gene.The disclosure also provides a method of evaluating the efficacy of atreatment for an inherited neuropathy in a subject, the methodcomprising administering to the subject an agent selected from the groupconsisting of an aldose reductase inhibitor (e.g., Alrestatin,Epalrestat, Diepalrestat, Fidarestat, Imirestat, Lidorestat,Minalrestat, Ponalrestat, Ranirestat, Salfredin B₁₁, Sorbinil,Tolrestat, Zenarestat, and/or Zopolrestat), an aldose reductaseantisense oligonucleotide, a polynucleotide that encodes a SORD peptide,a SORD peptide, an agent that blocks expression of a mutant SORD gene,and an agent that corrects the mutation in SORD gene (or a combinationof any of the foregoing); and measuring the level of sorbitol in asubject.

It is understood that each feature or embodiment, or combination,described herein is a non-limiting, illustrative example of any of theaspects of the disclosure and, as such, is meant to be combinable withany other feature or embodiment, or combination, described herein. Forexample, where features are described with language such as “oneembodiment,” “some embodiments,” “various embodiments,” “relatedembodiments,” each of these types of embodiments is a non-limitingexample of a feature that is intended to be combined with any otherfeature, or combination of features, described herein without having tolist every possible combination. Such features or combinations offeatures apply to any of the aspects of the invention.

The headings herein are for the convenience of the reader and notintended to be limiting. Additional aspects, embodiments, and variationsof the invention will be apparent from the Detailed Description and/ordrawings and/or claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F. SORD gene and pedigrees. Biallelic mutation in SORD causeautosomal recessive dHMN/CMT2. (FIG. 1A) Representative pedigrees ofdHMN/CMT2 families carrying biallelic mutations in SORD. The squaresindicate males and the circles females. The diagonal lines are used fordeceased individuals. Patients are indicated with filled shapes. (FIG.1B) Schematic diagram showing all exons, introns and untranslatedregions (UTRs) of SORD on the basis of NCBI Reference Sequence: NM003104.6. The gray and white boxes represent the coding sequence andUTRs of SORD, respectively. Variants identified in the familiesconsidered in the present study map throughout the coding region of thegene. The nonsense c.757delG; p.(Ala253GlnfsTer27) variant on exon 7,was identified at particular high frequency. (FIG. 1C) Distribution ofmutation across SORD protein domains. (FIG. 1D) SORD protein orthologsalignments showing that the four missense substitutions identified indHMN/CMT2 families in this study are located at highly conservedresidues across species from humans to elephants (FIGS. 1E and 1F)Magnification the nucleotide sequence of a highly homologous region inexon 7 in SORD (reverse strand) and SORD2P (forward strand). Nucleotidesdiffering in SORD2P from SORD are indicated with an arrow, including aFIG. 1C deletion in SORD2P. Representative electropherograms shows thatin SORD the c.757delG; p.(Ala253GlnfsTer27) variant found in homozygousstate in dHMN/CMT2 patients and heterozygous state in available patents(right box, upper plot) is absent in biallelic state from healthycontrols (right box, lower plot), but it is fixated in SORP2P (left box,lower plot).

FIGS. 2A-C. Decreased SORD expression and sorbitol accumulation inpatients fibroblasts. (FIG. 2A) Schematic representation of the two-steppolyol pathway converting glucose to fructose. (FIG. 2B) Immunoblotshowing protein level of SORD using the polyclonal antibody ab189248 andnormalized to Tubulin in healthy control (n=4, lane 1-4), heterozygouscarriers of c.757delG; p.(Ala253GlnfsTer27) variant in SORD (n=2, lane10-11) and patients carrying homozygous c.757delG; p.(Ala253GlnfsTer27)change (n=4, lane 5-8) or compound heterozygous c.757delG;p.(Ala253GlnfsTer27) variant together with a second nonsense c.895C>T;p.(Arg299Ter) mutation (n=1, lane 9). (FIG. 2C) Levels of intracellularsorbitol as measured by UPLC and normalised to protein content inhealthy controls (n=5) and patients carrying biallelic nonsensemutations in SORD (n=5). The graphs show the mean±s.d. and datadistribution (dots). A two-tailed t-test was performed to compare SORDencoded protein (FIG. 2B) or sorbitol level (FIG. 2C) across groups.Statistical significance is indicated as *, ** or *** if P-value <0.05,<0.01 or <0.001, respectively. All experiments were repeatedindependently twice with similar results.

FIGS. 3A-F. Loss of Drosophila Sord2 causes age-dependent synapticdegeneration. (FIG. 3A) 3D structure of Drosophila visual system showingthe lamina, medulla, and lobula. The xy- and xz-planes showing thephotoreceptor terminals and lamina neurons are indicated. (FIG. 3B)Lamina of yw control fly at 2 DAE. The organized lamina cartridges andcolumnar photoreceptor neurons are shown in the xy-plane and xz-plane,respectively. (FIG. 3C) Laminae of Sodh2^(MB01265/MB01265) homozygousflies at 2 DAE and 10 DAE. Arrowheads indicate the lamina vacuoles.Boxes indicate higher magnification areas of the lamina. The intensityof BRP is indicated. Dotted lines indicate the area of lamina vacuoles.Scale bar: 30 μm. (FIG. 3D) Quantification of the vacuole number, size,and BRP intensity. A total of 3 laminae of each group were quantified.Data are presented as mean±s.d. Statistical analysis was performed usingTwo-Way ANOVA followed by post-hoc Tukey's multiple comparison test.*P<0.05, **P<0.01, ****P<0.0001. (FIGS. 3E-3F) Locomotor activity ofcontrol flies (yw) and Sodh2^(MB01265/MB01265) (FIG. 3E) or Sodh1 andSodh2 pan-neuronal double knockdown (RNAi) (FIG. 3F) flies. n=10 in eachgroup. Data are presented as mean±s.d. Statistical analysis wasperformed using Two-Way ANOVA followed by post-hoc Tukey's multiplecomparison test. ****P<0.0001

FIGS. 4A-G. Treatment with aldose reductase inhibitors Epalrestat andRanirestat decrease sorbitol level and restore function. (FIG. 4A)Intracellular sorbitol level as measured by UPLC and normalised toprotein content in fibroblasts from healthy controls (n=5, circle dots)and patients carrying biallelic nonsense mutations in SORD (n=5, squaredots) after three days of treatment with Epalrestat 100 μM, Ranirestat10 μM or DMSO. (FIG. 4B) Sorbitol level as measured by UPLC frombrain/head homogenates and normalised to protein concentration fromwild-type (yw, empty circle dots), Sodh2^(MB01265/MB01265) (full circledots) and neuron-specific knock-down of Sodh1 and Sodh1 by RNAi (squaredots) Drosophilae at 10 days after eggs enclosure. Sodh2 Mimic and Sodh1and Soh2 RNAi Drosophilae were fed with either 80 μM Epalrestat, 80 μMRanirestat or DMSO. The graphs show the mean±s.d. A two-tailed t-testwas performed to compare sorbitol level. Statistical significance isindicated as *, ** or *** if P-value <0.05, <0.01 or <0.001,respectively, unless otherwise specified. All experiments were repeatedindependently twice with similar results. (FIG. 4C) Locomotor activityof control flies (yw) feeding with DMSO, Sodh2^(MB01265/MB01265) fliesfeeding with DMSO, 80 μM Epalrestat, or 80 μM Ranirestat (n=10 in eachgroup). Data are presented as mean±s.d. Statistical analysis wasperformed using Two-Way ANOVA followed by post-hoc Tukey's multiplecomparison test. * P<0.05, *** P<0.001 (FIGS. 4D-4F) Laminae ofSodh2^(MB01265/MB01265) homozygous flies at 10 DAE and 40 DAE fed withDMSO (FIG. 4D), 80 μM Epalrestat (FIG. 4E), or 80 μM Ranirestat (FIG.4F). Arrowheads indicate the lamina vacuoles. Boxes indicate highermagnification areas of the lamina. The intensity of BRP is indicated.Dotted lines indicate the area of lamina vacuoles. Scale bar: 30 μm.(FIG. 4G) Quantification of the vacuole number, size, and BRP intensityof (FIGS. 4D-4F). n=3. Data are presented as mean±s.d. Statisticalanalysis was performed using Two-Way ANOVA followed by post-hoc Tukey'smultiple comparison test. *P<0.05, **P<0.01, ****P<0.0001.

FIG. 5. Pedigrees of families carrying biallelic mutations in SORD. Thesquares indicate males and the circles females. The diagonal lines areused for deceased individuals. Patients are indicated with filledshapes.

FIGS. 6A-B. Double knockdown of Drosophila Sodh1 and Sodh2 lead toage-dependent synaptic degeneration. (FIG. 6A) Laminae of Sodh1 andSodh2 double knockdown homozygous flies at 2 DAE and 10 DAE. Arrowheadsindicate the lamina vacuoles. Boxes indicate higher magnification areasof the lamina. The intensity of BRP is indicated. Dotted lines indicatethe area of lamina vacuoles. Scale bar: 30 μm. (FIG. 6B) Quantificationof the vacuole number, size, and BRP intensity. A total of 3 laminae ofeach group were quantified. Data are presented as mean±s.d. Statisticalanalysis was performed using Two-Way ANOVA followed by post-hoc Tukey'smultiple comparison test. *P<0.05, **P<0.01, ****P<0.0001.

FIG. 7. Treatment with aldose reductase inhibitors Epalrestat andRanirestat restore locomotor function in Sodh1 and Sodh2 doubleknockdown flies. Locomotor activity of control flies (yw) feeding withDMSO (dots, first data point from the left for each DAE pointindicated), or flies with neuronal specific knockdown of Sodh1 and Sodh2feeding with DMSO (squares, second data point from the left for each DAEpoint indicated), 80 μM Epalrestat (squares, third data point from theleft for each DAE point indicated), or 80 μM Ranirestat (squares, forthdata point from the left for each DAE point indicated). n=10 in eachgroup. Data are presented as mean±s.d. Statistical analysis wasperformed using Two-Way ANOVA followed by post-hoc Tukey's multiplecomparison test. ***P<0.001, ****P<0.0001.

FIG. 8. An illustration of an exemplary expression vector encoding theSORD peptide (pAAV-SORD).

FIG. 9. An exemplary complete AAV vector DNA sequence including the SORDcoding sequence (pAAV-SORD) (SEQ ID NO: 1).

FIG. 10. SORD primer sequences and thermocycling conditions. PCR:polymerase chain reaction; Fw: forward; Rv: reverse.

FIG. 11. Clinical features of patients with hereditary neuropathy andcarrying biallelic mutations in SORD.

FIG. 12. Clinical features of patients affected by hereditary neuropathyand carrying the biallelic mutations in SORD. Categorical data areexpressed as N (%) if data is available in all individuals or N/numberindividuals considered (%). Continuous variables are expressed asmean±standard deviation (min-max). CMT, Charcot-Marie-Tooth, dHMN,distal hereditary motor neuropathy.

FIG. 13. Fasting sorbitol level in serum from ten unrelated healthycontrols and ten patients carrying biallelic p.Ala253GlnfsTer27mutations in SORD. The graphs show the mean s.d. and data distribution(dots), and the p-value of two-tailed t-tests comparing SORD protein andsorbitol levels across groups—* p<0.05, ** p<0.01, and *** p<0.001. Allexperiments were twice repeated independently.

FIGS. 14A-14C. Exemplary vector design for SORD gene replacementtherapy. (FIG. 14A) AAV-9 packaged vector design for a SORD genereplacement therapy. CB7 promotors have been shown to be effective indriving high expression, followed by the SORD cDNA (NCBI ReferenceSequence: NM 003104.6), a Posttranscriptional Regulatory Element (WPRE)to further enhance expression and target specificity, and thetranscription termination poly(A) element. Further origin of replication(pUC-ori) and ITR sequences (inverted terminal repeat). (FIG. 14B) SORDcDNA sequence. (FIG. 14C) SORD polypeptide sequence.

FIGS. 15A-15D. Significant knock-down of aldose reductase (AR) (AKR1B1gene) via an antisense oligonucleotide (ASO) (AR 1A, (SEQ ID NO: 22)).Targeting ASO (AR 1A) sequence and ASO-S scrambled sequence (AR-S 1A,(SEQ ID NO: 47)) are shown in FIG. 15A.

FIG. 15B shows the modifications to the nucleotide backbone of the ASOs.This was carried out in a SORD patient fibroblast and controlfibroblasts and normalized to β-tubulin and measured via Western blot(FIGS. 15C-15D). A further control is a scrambled version of the ASO-S(AR-S 1A) exhibiting random nucleotides was used (FIG. 15C).

FIG. 16. A table of antisense oligonucleotide sequences and target sitesin Homo sapiens aldo-keto reductase family 1 member B (AKR1B1), exontargets only. Filter criteria: A) 40%<=GC %<=60%; B) Antisense oligobinding energy <=−8 kcal/mol; C) No GGGG in the target sequence.

FIG. 17. A table of antisense oligonucleotide (ASO) sequences an targetsites in Homo sapiens aldo-keto reductase family 1 member B (AKR1B1),exon targets only. Filter criteria: A) 40%<=GC %<=60%; B) No GGGG in thetarget sequence; C) Average unpaired probability for target sitenucleotides >=0.5; D) For each peak in the accessibility profile that isabove the threshold probability of 0.5, all sites targeted to this samepeak are ranked by their average unpaired probability (the higher thebetter) and at most n sites are selected for each peak, where n isdetermined by max([width of peak/site length], 2); E) Among sitessatisfying criteria A-D, the top 20 unique ones with the highest averageunpaired probability are listed.

FIG. 18. A table of antisense oligonucleotide (ASO) sequences and targetsites in Homo sapiens aldo-keto reductase family 1 member B (AKR1B1),hg19_dna range=chr7:134127102-134143944 (intronic targets only). Filtercriteria: A) 40%<=GC %<=60%; B) No GGGG in the target sequence; C)Average unpaired probability for target site nucleotides >=0.5; D) Foreach peak in the accessibility profile that is above the thresholdprobability of 0.5, all sites targeted to this same peak are ranked bytheir average unpaired probability (the higher the better) and at most nsites are selected for each peak, where n is determined by max([width ofpeak/site length], 2); E) Among sites satisfying criteria A-D, the top20 unique ones with the highest average unpaired probability are listed.

DETAILED DESCRIPTION

The disclosure provides a method of detecting and/or treating inheritedneuropathy and related inherited conditions.

Inherited (or hereditary) neuropathies include, but are not limited toCharcot-Marie-Tooth disease (CMT), hereditary motor and sensoryneuropathy, hereditary motor neuropathy, distal hereditary motorneuropathy (dHMN), axonal neuropathies, intermediate neuropathies, andamyotrophic lateral sclerosis type ALS4.

In various aspects, the disclosure provides a method wherein thepresence of a mutation in the sorbitol dehydrogenase (SORD) gene isdetected in a sample from a subject. The mutation may be detected byexamining the DNA sequence of the gene, examining RNA, or examiningproteins with mutations that result in some loss of function.

Disclosed herein is the identification of biallelic mutations in theSorbitol dehydrogenase gene (SORD) associated with the most frequentrecessive form of CMT. SORD encodes sorbitol dehydrogenase, an enzymewhich converts sorbitol to fructose. It belongs to the two-step polyolpathway previously identified as pivotal to nerve damage inhyperglycemic condition of diabetes. Forty-two cases of CMT acrossdifferent ethnicities were identified as carrying a nonsense mutation inSORD, c.757delG; p.Ala253GlnfsTer27, either in homozygous or compoundheterozygous state. By screening the p.Ala253GlnfsTer27 change inadditional cases and multiple control sets, this variant was establishedas one of the most common pathogenic alleles in men inherited accordingto Mendel's law (MAF=0.003). Patient fibroblast cultures exhibit acomplete loss of SORD protein as well as loss of intracellular sorbitolaccumulation, which causes tissue damage. Loss of Sodh1 in Drosophilaled to synaptic degeneration and progressive motor impairment. Notably,reduction of polyol influx by treatment with aldose reductase inhibitorsfully rescued intracellular sorbitol levels in patient fibroblasts and aSodh1 Drosophila model. In the latter model, the treatment alsocompletely ameliorated motor and eye phenotypes. Together, thesefindings demonstrate a major role of the polyol pathway and sorbitolaccumulation in hereditary neuropathies and establish the molecularcause for a potentially treatable condition in a significant fraction ofcases. These findings also represent an example of convergingpathomechanisms of hereditary and acquired neuropathies with a broaderimpact in the field of diabetes.

Thus, in various aspects of the disclosure, the method comprisesdetecting the SORD gene mutation 753delG; p.(Ala253GlnfsTer27),c.757delG; p.Ala253GlnfsTer27, c.28C>T; p.Leu10Phe, c.316_425+165del;p.Cys106Ter, c.329G>C; p.Arg110Pro, c.298C>T; p.Arg100Ter, c.295C>T;p.Arg299Ter, c.964G>A; p.Val322Ile, c.458C>A; p.Ala153Asp; a deletion ofindividual or multiple coding exons or the entire SORD gene via a copynumber variation; or any protein truncating mutation and/or mutationthat leads to a “loss of function” or a hypomorphic function of theprotein.

In various aspects, the SORD mutation is detected using DNA sequencingmethods such as whole exome sequencing, whole genome sequencing (WGS)and/or next-generation sequencing (NGS), allele specificoligonucleotides, polymerase chain reaction (PCR), quantitative orreal-time PCR (qPCR), multiplex PCR, nested PCR, AmplificationRefractory Mutation System (ARMS) PCR, Multiplex ligation-dependentprobe amplification (MLPA), Denaturing gradient gel electrophoresis(DGGE), Single-Strand Conformation Polymorphism (SSCP), ProteinTruncation Test (PTT), RFLP, DNA microarray, RNA-seq, using CRISPR-basedmutation detection (e.g., CRISPR-Chip, Hajian et al., Nature BiomedicalEngineering 3, 427-437 (2019)) or other DNA or RNA mutation detectionmethods suitable for mutation detection.

In various aspects, the SORD mutation is detected by examining proteinsusing western blotting (immunoblot), High-performance liquidchromatography (HPLC), Liquid chromatography-mass spectrometry (LC/MS),antibody dependent methods such as enzyme-linked immunosorbent assay(ELISA), protein immunoprecipitation, protein immunostaining, proteinchip methods or other protein detection methods suitable for mutationdetection.

Optionally, the method further comprises measuring sorbitol levels in asample of the subject. Methods of measuring sorbitol include, e.g.,enzymatic assays, fluorimetric assays, chromatography-based methods, andspectroscopy-based methods. An exemplary method of sorbitol measurementis provided in the Examples.

The disclosure further provides a method of characterizing a neuropathy(e.g., inherited neuropathy) and related conditions involving a SORDmutation. In various aspects, the method comprises measuring sorbitollevels in a biological sample of a subject suffering from a neuropathy.In various aspects, the method comprises detecting increased levels ofsorbitol in the biological sample. By “increased levels of sorbitol” ismeant, e.g., sorbitol levels above about 10 mg/L. SORD-relatedneuropathy leads to high levels of sorbitol in patients, as described inthe Examples and FIG. 13. As such, detection of sorbitol levels aboveabout 10 mg/L indicates that the neuropathy is an inherited neuropathyassociated with a SORD mutation, thereby allowing a clinician tocharacterize the neuropathy afflicting the subject. Optionally, themethod comprises a treatment step comprising administering to thesubject an agent selected from the group consisting of an aldosereductase inhibitor; an aldose reductase antisense oligonucleotide; apolynucleotide that encodes a SORD peptide; a SORD peptide; an agentthat blocks expression of a mutant SORD gene; and an agent that correctsthe mutation in SORD gene.

In various aspects, the disclosure provides a method comprisingidentifying a mutation in the sorbitol dehydrogenase (SORD) gene in asample from a subject before or after a step of measuring sorbitollevels in the subject. In this regard, the method may be used to confirma diagnosis of inherited neuropathy. Similarly, the disclosure providesa method for identifying a SORD mutation that is pathogenic, the methodcomprising measuring sorbitol levels in a subject comprising a mutationin the SORD gene. The presence of increased sorbitol levels (e.g.,greater than about 10 mg/L) indicates that the SORD mutation ispathogenic.

Alternatively (or in addition), the method may be used to evaluate theefficacy of a treatment for an inherited neuropathy in a subject. Inthis regard, the method comprises administering a therapy to thesubject, then measuring sorbitol levels in a biological sample. Adecrease in sorbitol levels compared to the level of sorbitol observedpre-treatment (e.g., a reduction of sorbitol levels below about 10 g/L)indicates an improvement in the subject's condition. The materials andmethods described herein may also characterize patient compliance intaking medication for treatment of SORD-related inherited neuropathiesor monitor the success of candidate therapeutics in clinical trials.

The sample may be any biological sample taken from the subject,including, but not limited to, any tissue, cell, or fluid (e.g., blood,plasma, serum, or urine) which can be analyzed for a trait of interest,such as the presence or amount of a nucleic acid (e.g., SORD mRNA), aprotein (e.g., SORD protein), or sorbitol. In various embodiments, thebiological sample is a plasma, serum, saliva, urine, or skin sample.

A “subject” as referred to herein, can be any mammal, such as humans.Animals of agricultural importance, such as bovine, equine, and porcineanimals, are contemplated, as well as animals important as domesticpets, including canines and felines; animals important in research,including rodents and primates; and large endangered species and zooanimals such as primates, felines, giraffes, elephants, rhinos.

In various aspects, the method comprises treating the subject byadministering to the subject a composition that comprises one or morealdose reductase inhibitors. In some embodiments, the aldose reductaseinhibitor is Alrestatin, Epalrestat, Diepalrestat, Fidarestat,Imirestat, Lidorestat, Minalrestat, Ponalrestat, Ranirestat, SalfredinB₁₁, Sorbinil, Tolrestat, Zenarestat, or Zopolrestat. Aldose reductaseinhibitors are reviewed in Expert Opin Ther Pat. 2019; 29(3):199-213;Chatzopoulou et al., Expert Opin Ther Pat. 2012; 22(11):1303-23(incorporated by reference in their entirety).

In some embodiments, enzyme replacement therapy is employed, and a SORDpeptide is administered to the subject. As such, the therapy supplementsSORD peptide levels where endogenous SORD levels are inadequate orabsent. An exemplary SORD peptide is provided in SEQ ID NO: 46. Thedisclosure contemplates use of a peptide that comprises at least 80%, atleast 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO:46.

In various embodiments, the method comprises administering to thesubject a polynucleotide (e.g., an aldose reductase anti senseoligonucleotide, a polynucleotide that encodes the SORD peptide/protein,an agent that blocks expression of a mutant SORD gene, and/or an agentthat corrects the mutation in SORD gene). Polynucleotides are typicallydelivered to a host cell via an expression vector, which includes theregulatory sequences necessary for delivery and expression, although useof expression vectors are not required in the context of the disclosure.In some aspects, the constructs described herein include a promoter(e.g., cytomegalovirus (CMV) promoter or CB7 promoter), a protein codingregion (optionally with non-coding (e.g. 3′-UTR) regions that facilitateexpression), transcription termination sequences, and/or regulatorelements sequences (e.g., Posttranscriptional Regulatory Element (WPRE),poly(A) element, origin of replication (pUC-ori) and/or ITR sequences(inverted terminal repeat)). In various aspects, the constructsdescribed herein include one or more of vector features listed inTable 1. Vector features are also reviewed in Powell et al., Discov Med.2015; 19(102): 49-57 (incorporated by reference in its entirety). Forexample, the Cre-loxP system may be utilized to express a peptide ofinterest (e.g., a SORD peptides, optionally in a specific tissue ofinterest). Expression vectors may be viral-based (e.g., retrovirus-,adenovirus-, or adeno-associated virus-based) or non-viral vectors(e.g., plasmids). Non-vector based methods (e.g., using naked DNA, DNAcomplexes, etc.) also may be employed. Optionally, the vector is a viralvector, such as a lentiviral vector or baculoviral vector, and invarious preferred embodiments the vector is an adeno-associated viralvector (AAV). The expression vector may be based on any AAV serotype,including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9,AAV-10, AAV-11, AAV-12, or AAV-13. Polynucleotides also may be deliveredvia liposomes, nanoparticles, exosomes, microvesicles,hydrodynamic-based gene delivery, or via a “gene-gun.”

TABLE 1 Vector feature elements Post- transcrip- Polyadeny- NeuronalIntrons tional lation Ubiquitous specific (enhanced regulatory signalBacterial Promoters promoter expression) elements enhancers resistanceCMV, NFL, b-Glob, HPRE, hGH, Ampicillin Cba, NFH, MVM, I.IX, WPRE bGHpA,Kanamycin CAG, synapsin, adenovirus SPA, CBh, CaMKII, SD/ SV40 lateEF1-α, Hb9, immunoglob- PGK, MeCP2 ulin SA, UBC SV40 late SD/SA

Titers of AAV to be administered in methods of the disclosure will varydepending, for example, on the particular AAV, the mode ofadministration, the treatment goal, the individual, and the cell type(s)being targeted, and may be determined by methods known in the art.Titers of AAV may range from about 1×10⁶, about 1×10⁷, about 1×10⁸,about 1×10⁹, about 1×10¹⁰, about 1×10¹¹, about 1×10¹², about 1×10¹³ toabout 1×10¹⁴ or more DNase resistant particles (DRP) per ml. Dosages mayalso be expressed in units of viral genomes (vg).

In various embodiments, a polynucleotide that encodes a SORD peptide isadministered to the subject. The amino acid sequence of SORD is providedas SEQ ID NO: 46 (FIG. 14C, NCBI Reference Sequence: NP 003095.2). Thepolynucleotide used in the method optionally encodes the amino acidsequence of SEQ ID NO: 46 or a sequence that is at least 80%, at least85%, at least 90%, at least 95%, or 100% identical to the amino acidsequence of SEQ ID NO: 46 (which retains the function of SORD).Optionally, the polynucleotide comprises SEQ ID NO: 45 (FIG. 14B) or asequence that is at least 80%, at least 85%, at least 90%, at least 95%,or 100% identical to the polynucleotide sequence of SEQ ID NO: 45 (andwhich encodes SORD). Exemplary expression vectors comprising apolynucleotide encoding the SORD peptide are illustrated in FIGS. 8 and14A. The polynucleotide, in at least one aspect of the disclosure,comprises the nucleic acid sequence shown in FIG. 9 (SEQ ID NO: 1),which corresponds to the sequence of an AAV vector comprising apolynucleotide encoding SORD.

In various embodiments, the method comprises administering to thesubject an agent that blocks expression of a mutant SORD gene. An agentthat blocks expression of a mutant SORD gene refers to an agent thatinterferes with expression of a SORD gene so that SORD gene expressionand/or SORD protein levels are reduced compared to basal/wild-typelevels. It will be appreciated that “blocking” expression of a mutantSORD gene does not require 100% abolition of expression and SORDproduction; any level of reduced expression of aberrant SORD may bebeneficial to a subject. Exemplary agents include, but are not limitedto, antisense oligonucleotides (ASO), short hairpin RNA (shRNA), smallinterfering RNA (siRNA), or micro RNA (miRNA).

In various embodiments, the method comprises administering to thesubject an aldose reductase antisense oligonucleotide which targets thealdose reductase sequence such that expression of the enzyme is blocked.An aldose reductase, aldo-keto reductase family 1 member B (AKR1B1), isencoded by SEQ ID NO: 48 (NCBI Reference Sequence: NM 001628). An aldosereductase antisense oligonucleotide interferes with expression of analdose reductase gene (AKR1B1), so that AKR1B1 gene expression and/oraldose reductase protein levels are reduced compared to basal/wild-typelevels. It will be appreciated that “blocking” expression of an aldosereductase gene (the AKR1B1 gene) does not require 100% abolition ofexpression and aldose reductase production; any level of reducedexpression of aldose reductase may be beneficial to a subject. Forexample, in various aspects, the aldose reductase antisenseoligonucleotide that reduces the expression of aldose reductase. An ASOis a single-stranded deoxyribonucleotide, which is complementary to anmRNA target sequence. In various aspects, the aldose reductase antisenseoligonucleotide targets an exonic or intronic sequence of the aldosereductase gene.

In an exemplary method for identifying ASO sequences targeting aldosereductase, the following criteria were used: A) sequences targetingaldose reductase (AKR1B1) were selected which contained <=40% GC or<=60% GC content; B) sequences containing GGGG nucleotides wereexcluded; C) sequences with an average unpaired probability for targetsite nucleotides >=0.5 were selected; D) for each peak in theaccessibility profile that was above the threshold probability of 0.5,all sites targeted to the same peak were ranked by their averageunpaired probability (the higher the better) and at most n sites areselected for each peak, where n is determined by max width of peak/sitelength. Exemplary agents satisfying these criteria are provided in Table2. Additional exemplary ASO sequences and filter criteria are shown inFIGS. 16-18.

TABLE 2 ASO sequences targeting AKR1B1/aldose reductase. Starting EndingSEQ SEQ Average Binding target target Target ID antisense ID GC unpairedsite position position Seq (5′-3′) NO  oligo (5′-3′) NO contentprobability disruption  475 AGGUGGAGAUGAUCUUAAAC 21 GTTTAAGATCATCTCCACCT22 40.00% 0.594 12.5  476  495 GGUGGAGAUGAUCUUAAACA 23TGTTTAAGATCATCTCCACC 24 40.00% 0.592 12.6  484  503 UGAUCUUAAACAAACCUGGC25 GCCAGGTTTGTTTAAGATCA 26 40.00% 0.66 12.5  490  509UAAACAAACCUGGCUUGAAG 27 CTTCAAGCCAGGTTTGTTTA 28 40.00% 0.633  9.2  534 555 CAGGUGGAGAUGAUCUUAAACA 29 TGTTTAAGATCATCTCCACCTG 30 40.90% 0.58412.6  545  566 GAUCUUAAACAAACCUGGCUUG 31 CAAGCCAGGTTTGTTTAAGATC 3240.90% 0.611 10.3  548  569 CUUAAACAAACCUGGCUUGAAG 33CTTCAAGCCAGGTTTGTTTAAG 34 40.90% 0.646  9.5  567  588AAGUAUAAGCCUGCAGUUAACC 35 GGTTAACTGCAGGCTTATACTT 36 40.90% 0.584  7.7 739  760 UCAAGGCGAUCGCAGCCAAGCA 37 TGCTTGGCTGCGATCGCCTTGA 38 59.10%0.669 10  741  762 AAGGCGAUCGCAGCCAAGCACA 39 TGTGCTTGGCTGCGATCGCCTT 4059.10% 0.694  9.3 1025 1046 ACCUGUGUUUCUUGCCUCAUUU 41AAATGAGGCAAGAAACACAGGT 42 40.90% 0.641  5.5

In various embodiments, the nucleotide backbone of ASO sequences aremodified to a chimeric or gapmer design to reduce gene expression whencompared to basal/wild-type levels. In various embodiments, a gapmerdesign requires a designation of 3-5 nucleotides on each end of theantisense oligonucleotide sequence to harbor modifications in the ribosesugar moiety resistant to RNase H recognition and other nucleases, whileall other nucleotides contain an RNase H compatible modification. RNaseH is responsible for cleaving RNA-DNA duplexes such as those formedbetween aberrant mRNA transcripts and synthetically designed DNAantisense oligonucleotides. In various embodiments, the modification tothe ASO sequences includes, but is not limited to Phosphorothioate(PS)—RNase H recognizable, phosphorodiamidate morpholino (PMO)—RNase Hresistant, 2′-O-methyl—RNase H resistant, 2′-O-methoxyethyl (MOE)—RNaseH resistant, locked Nucleic Acid (LNA)—RNase H resistant,ethylene-bridged nucleic acid (ENA)—RNase H resistant, or(S)-constrained ethyl (cEt)—RNase H resistant. Exemplary modification ofan ASO sequence is shown in FIGS. 15A-15B. Modifications to ASOsequences are reviewed in Scoles et al., Neurol Genet. 2019; 5(2):e323.(incorporated by reference in its entirety).

In various aspects, the method employs RNA interference (RNAi) toregulate expression of SORD. The RNAi pathway is summarized in Duan(Ed.), Section 7.3 of Chapter 7 in Muscle Gene Therapy, SpringerScience+Business Media, LLC (2010). Suitable agents include, e.g.,siRNA, miRNA, and shRNA. A shRNA/Hairpin Vector is an artificial RNAmolecule (nucleotide) with a tight hairpin turn that can be used tosilence target gene expression via RNAi. shRNA is an advantageousmediator of RNAi in that it has a relatively low rate of degradation andturnover, but it often requires use of an expression vector. Inexemplary aspects, the disclosure includes the production andadministration of an AAV vector expressing one or more shRNAs targetingSORD. The expression of shRNAs is regulated by the use of variouspromoters. In various aspects, polymerase II promoters, such as U6 andH1, and polymerase III promoters are used. In some aspects, U6 shRNAsare used. It will be appreciated that RNAi also may be used todownregulate (i.e., block) expression of aldose reductase (e.g.,AKR1B1); as such, the disclosure contemplates sue of siRNA, miRNA, andshRNA which targets aldose reductase intronic or extronic sequences toblock the expression of aldose reductase.

Traditional small/short hairpin RNA (shRNA) sequences are usuallytranscribed inside the cell nucleus from a vector containing a Pol IIIpromoter such as U6. The endogenous U6 promoter normally controlsexpression of the U6 RNA, a small RNA involved in splicing, and has beenwell-characterized (Kunkel et al., Nature. 322(6074):73-7 (1986); Kunkelet al., Genes Dev. 2(2):196-204 (1988); Paule et al., Nucleic Acids Res.28(6):1283-98 (2000)). The disclosure includes both murine and human U6or H1 promoters. The shRNA containing the sense and antisense sequencesfrom a target gene connected by a loop is transported from the nucleusinto the cytoplasm where Dicer processes it into siRNAs.

In some aspects of the disclosure, an agent that corrects the mutationin the SORD gene is employed. An agent that corrects the mutation inSORD gene refers to an agent capable of modifying the SORD codingsequence or a regulatory element and/or non-coding region associatedwith the SORD gene to achieve a desired change in the sequence. Invarious aspects, genome editing may be used to replace part or all ofthe SORD gene sequence or alter SORD protein expression levels. Invarious embodiments, the agent may comprise components employed ingenome-editing techniques, such as designer zinc fingers, transcriptionactivator-like effectors nucleases (TALENs), or CRISPR-Cas (clusteredregularly interspaced short palindromic repeats-CRISPR associated)systems. An exemplary agent for use in the method of the disclosure is,DNA encoding Cas9 molecules and/or gRNA molecules. Cas9 and gRNA can bepresent in a single expression vector or separate expression vectors.Adenoviral delivery of the CRISPR/Cas9 system is described in Holkers etal., Nature Methods (2014), 11(10):1051-1057 which is incorporated byreference in its entirety.

Other publications describing the CRISPR systems and Cas9 include thefollowing: Cong et al. Science (2013) 339:819-23; Jinek et al., Elife.(2013) 2:e00471; Lei et al. Cell (2013) 152: 1173-1183; Gilbert et al.Cell (2013) 154:442-51; Lei et al. Elife (2014) 3:e04766; Perez-Pinelaet al. Nat Methods (2013) 10: 973-976; Maider et al. Nature Methods(2013) 10, 977-979; U.S. Pat. Nos. 8,697,359; 8,771,945; 8,795,965;8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814;8,945,839; 8,993,233; 8,999,641; U.S. Application Publication No.2014/0068797; and International Patent Publication No. WO 2014/197568,all incorporated by reference in their entirety.

In some embodiments, CRISPR/Cas9 multiplexing may be used to targetmultiple genomic loci wherein two or more guide RNAs are expressed asdescribed in CRISPR 101: A Desktop Resource (1^(st) Edition), Addgene,January 2016 which is incorporated by reference in its entirety.

The terms “treating” or “treatment” refer to reducing or amelioratinginherited neuropathy and/or associated disorders and/or symptomsassociated therewith. These terms include reducing or delaying thefrequency of occurrence or recurrence of the neuropathy or symptomsassociated therewith (i.e., lengthening the period of remission in apatient who had suffered from the disorder), as well as reducing theseverity of the disorder or any symptoms associated therewith. It isappreciated that, although not precluded, “treating” or “treatment” of adisorder or condition does not require that the disorder, condition, orsymptoms associated therewith be completely eliminated.

A dose of an active agent (e.g., an aldose reductase inhibitor, analdose reductase antisense oligonucleotide, a polynucleotide thatencodes a SORD peptide, a SORD peptide, an agent that blocks expressionof a mutant SORD gene, or an agent that corrects the mutation in SORDgene) will depend on factors such as route of administration (e.g.,local vs. systemic), patient characteristics (e.g., gender, weight,health, side effects), the nature and extent of the inherited neuropathyor associated disorder, and the particular active agent or combinationof active agents selected for administration.

The active agents described herein are provided in a composition (e.g.,a pharmaceutically-acceptable composition) which may contain formulationcomponents suitable for administration to a subject, as well asadditional therapeutic agents. Suitable methods of administering aphysiologically-acceptable composition, such as a pharmaceuticalcomposition comprising an agent described herein, are well known in theart. In various aspects, more than one route can be used to administerone or more of the agents disclosed herein. A particular route canprovide a more immediate and more effective reaction than another route.For example, in certain circumstances, it will be desirable to deliverthe composition orally; through injection or infusion by intravenous,intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, intralesional, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, urethral, vaginal, or rectalmeans; by controlled, delayed, sustained or otherwise modified releasesystems; by implantation devices; using nanoparticles; or as aconjugate.

It is contemplated the two or more active agents described herein may beadministered as part of a therapeutic regimen. Alternatively or inaddition, one or more of the active agents may be administered withother therapeutics as part of a therapeutic regimen. The active agent(s)may be administered as a monotherapy or as a combination therapy withother treatments administered simultaneously or metronomically. The term“simultaneous” or “simultaneously” refers to administration of twoagents within six hours or less (e.g., within three hours or within onehour each other). In this regard, multiple active (or therapeutic)agents may be administered the same composition or in separatecompositions provided within a short period of time (e.g., within 30minutes). The term “metronomically” means the administration ofdifferent agents at different times and at a frequency relative torepeat administration. Active agents need not be administered at thesame time or by the same route; preferably, in various embodiments,there is an overlap in the time period during which different activeagents are exerting their therapeutic effect. Additional aspects anddetails of the disclosure will be apparent from the following examples,which are intended to be illustrative rather than limiting.

EXAMPLES General Methods Families

All families provided written informed consent to participate in thestudy. The study protocol was approved by the institutional review boardof giving institutions. All patients were clinically evaluated byneurologists.

Whole Exome and Sanger Sequencing

Whole exome sequencing was performed in index individuals from sporadicand recessive CMT and dHMN families. The SureSelect Human All Exon 50 MBKit (Agilent) was used for in-solution enrichment, and the HiSeq 2500instrument (Illumina) was used to produce about 120 bp paired-endsequence reads. The Burrows-Wheeler aligner, and Freebayers were used toalign sequence reads and call variants. Final data were uploaded intoGENESIS software for analysis. A filtering approach to search forfamilies sharing the same homozygous variants were applied across allexomes in the database. Sanger sequencing, performed by EurofinsGenomics, confirmed segregation of the SORD variants. Polymerase chainreaction (PCR) was carried out in the Veriti Thermocycler (AppliedBiosystem) and Platinum Taq (ThermoFisher) was used to amplify theregions containing the target mutations. The following primers were usedto target specifically SORD and not SOR2P (FIG. 10).

Fibroblasts Culture

Fibroblasts were obtained from patients and cultured in Dulbecco'sModified Eagle Medium (ThermoFisher) supplemented with 10% fetal bovineserum (FBS), penicillin and streptomycin (Gibco). Cells were maintainedin 5% CO₂ at 37° C. in a humidified incubator. Asynchronous cellcultures were grown to approximately 80% confluency and treated withepalrestat (10004), ranirestat (10 μM) or DMSO for 72 hours. Mediacontaining the drugs or DMSO were changed every 24 hours.

Western Blot

Fibroblasts were lysed in RIPA buffer (ThermoFisher) containing proteaseinhibitor (Roche) and sonicated for 5 minutes with the Bioruptorsonication device (Diagenode). Cell lysates were centrifuged at 13,000×gfor 10 minutes at 4° C., and the supernatant was collected for proteinquantification (Pierce BCA Protein Assay Kit). 30 μg of protein samplewas mixed with Bolt LDS Sample Buffer and Sample Reducing Agent(ThermoFisher) and heated at 90° C. for 5 min. Samples were loaded onBolt 4-12% Bis-Tris Plus mini-gel followed by transfer into anitrocellulose membrane (Bio-Rad). Membrane was blocked with 5% non-fatmilk and incubated with anti-SORD (ab189248, Abcam) antibody for 2hours, washed with TB S containing 0.01% Tween 20 (Bio-Rad) andincubated with a secondary anti-rabbit antibody (Cell Signaling).Membrane was subsequently incubated with GAPDH primary antibody (SantaCruz) and secondary anti-mouse antibody (Cell Signaling).Chemoluminescence detection was performed with the SuperSignal West PicoPLUS Chemiluminescent Substrate and imaged with the FluorChem E(ProteinSimple).

Sorbitol Measurement

Fibroblasts were collected and lysed as described in Western blotsection in the absence of proteinase inhibitor. Sorbitol determinationin human fibroblast lysates was performed in ultra-performance liquidchromatography-tandem mass spectrometry (UPLC-MS/MS) (Waters AcquityUPLC & TQD mass spectrometer—Waters, Milford, Mass., USA). Fibroblastswere collected and lysed as described in Western blot section in theabsence of proteinase inhibitor. Proteinase inhibitor contains highconcentration mannitol, which is a sorbitol enantiomer, and caninterfere with UPLC-MS/MS sorbitol determination. Lysate samplesunderwent protein precipitation with Acetonitrile (1:5), ten-timedilution with Acetonitrile-water (50/50) and clean up on Oasis HLBcartridges (10 mg/1 ml), before injection in UPLC (3 μL). UPLCconditions: column, BEH Amide 1.7 μm (2.1×100 mm) at 88° C., eluent A,Acetonitrile 90%-water 5%-Isopropanol 5%, eluent B, Acetonitrile80%-water 20%, gradient elution 0 min., 100% A, 3.6 min. 100% B, flowrate, 0.45 ml/min. The retention time of sorbitol was 2.7 min. MS/MSconditions: interface, Electrospray interface in negative ion mode,Multiple Reaction Monitoring acquisition, m/z 180.9→88.9 (CV 24, CE 15).

For fasting sorbitol level testing, blood was collected after overnightfasting (last meal the evening before) in serum separator tubes. Sampleswere centrifuged at 500 g for 10 min. Serum was separated and frozenwithin an hour from blood collection. Sorbitol level was tested by UPLCusing a method adapted from Li et al. Biochem Biophys Res Commun. 2009Oct. 2; 387(4):778-83. Conditions were as follows: column, BEH Amide 1.7μm maintained at 25° C. (instead of 45° C.); eluent A, 10 mM ammoniumacetate pH10; eluent B, Acetonitrile. Flow rate, 0.6 ml/min with the 20same gradient. The retention time of Sorbitol was 6.0 min. MS/MSconditions were the same of fibroblast analysis. Serum samples underwentprotein precipitation with cold Methanol (1:5), five time dilution withAcetonitrile-water (50/50) and clean up on Oasis HLB cartridges (10 mg/1ml), before injection in UPLC (3 μL). Calibration curve was done inserum in sorbitol concentration range 0.1-20 mg/L.

Drosophila Stocks and Genetics

Unless specified, all flies were kept on cornmeal-molasses-yeast mediumat 25° C., 65% humidity, with 12 h light/12 h dark cycles. The followingfly strains used in this study were obtained from Bloomington DrosophilaStock Center: elav^(C155)-GAL4, GMR-GAL4, Sdh2^(MB01265) UAS-Sdh1RNAi,and UAS-Sdh2RNAi.

Drug Feeding

Epalrestat or ranirestat was dissolved in dimethyl sulfoxide (DMSO) toachieve a stock concentration of 10 mg/ml, and then mixed into 10 ml flyfood at a final concentration of 80 μg/ml. Equal amount of DMSO wasmixed into the fly food as control. The vials were dried at roomtemperature for 12 h before feeding.

Drosophila Lifespan Assay and Negative Geotaxis Assay

For lifespan assay, 100 newly enclosed female flies of each group werecollected and placed in vials of 20 individuals. Flies were transferredinto new vials every 2 days and the number of dead flies was counted.Survival data was plotted using Kaplan-Meier plot and compared betweengroups using log-rank test. For negative geotaxis behavior assay, 10age-matched female flies were placed in a vial marked with a black linedrawn horizontally 8 cm above the bottom. Flies were given 60 min tofully recover from CO₂ anesthesia, and were gently tapped onto thebottom and given 10 s to climb. Flies that crossed the 8 cm line werecounted. For each vial, this assay was repeated 10 times, and 10independent vials of each group (a total 100 flies per group) weretested. To minimize observer-expectancy bias, this assay was performedwith the examiner masked to the group assignment.

Drosophila Brain Dissection, Immunostaining, and Confocal Microscopy

Brain dissection and staining were carried out as previously described(Brazill et al., J Vis Exp. 2018; (138)). Briefly, fly brains weredissected in phosphate-buffered saline (PBS, pH 7.4), fixed in 4%formaldehyde for 10 min, and washed in PBTX (PBS containing 0.4% v/vTriton X-100) for 3 times (15 mins each). Brains were then incubatedwith primary mouse anti-BRP antibody (nc82, Developmental StudiesHybridoma Bank) at 1:250 dilution in 0.4% PBTX with 5% normal goat serumat 4° C. overnight with gentle shaking. After that, brains wereincubated with Cy3-conjugated anti-mouse secondary antibody (Rockland)and Cy5-conjugated anti-HRP (Jackson ImmunoLab) at 1:250 dilution at 4°C. overnight with gentle shaking, followed by4′,6-diamidino-2-phenylindole (DAPI, 1:300, Invitrogen) staining at roomtemperature for 10 min. Samples were mounted on glass slides withVECTASHIELD Antifade Mounting Medium (Vector Laboratories Inc.). Flybrain slides were imaged using an Olympus IX81 confocal microscope with60× oil immersion objective lens with a scan speed of 8.0 μs per pixeland spatial resolution of 1024×1024 pixels. Images were processed andanalyzed using FluoView 10-ASW (Olympus).

Additional aspects and details of the disclosure will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

Example 1: Identification of DNA Variants in CMT Using GENESIS Analysis

Inherited neuropathies, including Charcot-Marie-Tooth disease (CMT),represent an umbrella concept for clinically and geneticallyheterogeneous conditions affecting the peripheral nerves. CMT isclassified depending on conduction velocity as demyelinating (CMT1) andaxonal (CMT2) types. Distal hereditary motor neuropathy (dHMN)represents a form of CMT2 in which the burden of disease fallspredominantly or exclusively on motor nerves (Rossor, Tomaselli, andReilly 2016). As opposed to CMT1, for which over 90% of cases havemutations in known genes, only 20 to 30% of CMT2 and distal HMN patientsreceive a genetic diagnosis (Fridman et al. 2015). Since up to 70% ofCMT2 and dHMN cases are sporadic, it becomes more challenging toidentify candidate pathogenic genes from single case whole exome andgenomic sequences; therefore, large collective datasets are necessary.Using the data aggregation of over 1,100 CMT whole exome sequencing(WES) and whole genome sequencing (WGS) available at the GENESISanalysis platform provided the largest collection of such high qualitydata available (Gonzalez et al. 2015). Genes with significant DNAvariants present in multiple families were identified, as well asindividual alleles overrepresented in CMT cases. When querying a subsetof 598 undiagnosed CMT patients for recessive non-sense variants ingenes shared by >3 families and with minor allele frequency in thegnomAD control database of <1%, 12 cases were identified from 11unrelated families carrying a homozygous c.757delG; p.(Ala253GlnfsTer27)mutation in SORD. Four more cases from three unrelated families carriedthe heterozygous c.757delG; p.(Ala253GlnfsTer27) variant together with asecond variant, c.298C>T; p.(Arg100Ter) in family 2 c.329G>C;p.(Arg110Pro) in family 3, and c.458C>A; p.(Ala153Asp) in II-1 and 11-2of family 14 (FIG. 1A-D and FIG. 5). All mutations representedloss-of-function (LOF) alleles except c.329G>C; p.(Arg110Pro).Interestingly, the Arg110Pro change is adjacent to the previouslyreported Tyr111Phe (corresponding to Tyr110Phe in rat), which was shownto abolish SORD enzymatic activity and destabilize the protein (Hellgrenet al. 2007).

Interestingly, SORD has a non-functional highly homologous paralogue,the pseudogene SORD2P, which is thought to arise from the duplication ofSORD within a 0.5 Mb region on chromosome 15 (Carr et al. 2016) (FIG.1E). In order to specifically amplify SORD, but not SORD2P, in Sangerconfirmation studies, primers were designed that took advantage ofnucleotide sequence differences and distinct retrotransposon insertionsin both genic regions (FIG. 5). Notably, the c.757delG;p.(Ala253GlnfsTer27) mutation in exon 7 of SORD is fixated in thepseudogene SORP2P in over 95% of control chromosomes, along withnumerous additional exonic indel mutations, which prevent effectivetranslation of SOPR2P (1000 Genomes Project Consortium et al. 2015; Leket al. 2016). Because of the high similarity of the regions, a nestedPCR approach was necessary to obtain specific amplification of exon 7 ofSORD and distinguish it from the homologous region in SORD2P. Thepresence of the variants detected by WES was confirmed by Sangersequencing in all cases and segregation data in immediate relativecarriers was provided. (FIG. 1F and FIG. 5).

An independent set of 103 unresolved CMT2/dHMN cases WES at the (UCLInstitute of Neurology in London (UK)) were screened. Nine cases fromsix unrelated families were identified carrying the homozygousc.757delG; p.(Ala253GlnfsTer27) mutation in SORD (8.7%). A thirdindependent set of 297 recessive or sporadic CMT2/dHMN patients wasscreened by targeted Sanger sequencing of exon 7 of SORD, which wasextended to the other coding exons if one c.757delG;p.(Ala253GlnfsTer27) was identified, and revealed 20 additional cases(7%) from 18 families with biallelic mutations in SORD: 16 cases with ahomozygous c.757delG; p.(Ala253GlnfsTer27) mutation and four cases withc.757delG; p.(Ala253GlnfsTer27) in compound heterozygous state with asecond likely pathogenic variant. The latter included c.964G>A;p.(Val322Ile) in family 29, a 275 bp deletion c.316_425+165del in exon 4in family 30, a de novo c.28C>T; p.(Leu10Phe) in family 32, andc.895C>T; p.(Arg299Ter) in family 33. All changes have a minor allelefrequency (MAF) of <0.0001 in gnomAD (Lek et al. 2016). The residuesaffected by missense mutations are highly conserved across multiplespecies (FIG. 1D) with GERP scores greater than 3. Further, biallelicnon-sense variants in SORD were absent from 4,598 index cases affectedby distinct neurological disorders other than CMT present in the GENESISdatabase.

The allelic carrier frequency of the c.757delG; p.(Ala253GlnfsTer27)variant in the normal population is 0.003% based on an allelic count of94 out of 30,872 in gnomAD genomes (Lek et al. 2016). Of note, thegnomAD exome set detected the c.757delG; p.(Ala253GlnfsTer27) change ata significantly lower rate at MAF=0.00008, due to failure to pass randomforest filters. GENESIS uses the FreeBayes software for variant calling(Gonzalez et al. 2015), which may have resulted in an allele frequencycloser to the gnomAD genome based call set (MAF_(GENESIS)=0.002, 22 outof 9,196). Sanger sequencing of 600 healthy controls was performed,including 200 samples of European, 100 samples of Turkish and 200samples of Middle Eastern origin, and identified three heterozygous, butno homozygous, c.757delG; p.(Ala253GlnfsTer27) alleles (MAF=0.0025).These calculations support an estimated prevalence of the homozygousc.757delG; p.(Ala253GlnfsTer27) allele alone of ˜1/100,000 individuals,making it the most common individual pathogenic allele in axonalneuropathies and one of the most common alleles for any Mendeliandisease.

Overall 45 individuals affected by hereditary neuropathy from 38unrelated families were identified in the present study to carrybiallelic mutations in SORD (FIGS. 11 and 12). Of note, 71% of caseswere sporadic with no evidence of family history or consanguinity. Theformal clinical diagnosis was axonal CMT in 51% (n=16), distal HMN in40% (n=18), and intermediate CMT in 9% (n=4) of cases The mean age ofonset of the neuropathy was 17±8 years and walking difficulties was themost common complain at onset. Delayed motoric development milestoneswere uncommon, but two thirds of the patients reported foot deformities,indicating that the neuropathy probably started earlier in life. Atfirst examination all individuals had limb weakness, but only halfshowed sensory impairment. Weakness was mild in distal upper limbs andranged from mild to near complete paralysis in the distal lower limbs.Proximal muscles of the upper and lower limbs were typically unaffected.Seven patients had upper limb tremor, four had mild scoliosis and twohad mild hearing loss. One case had a concurrent and likely unrelatedsyndromic disorder encompassing dysmorphic features, non-progressivemental retardation since the age of three years, and spastic ataxia withevidence of cerebellar atrophy at brain MM. None of the patients hadcataract nor involvement of other organs. According to the CMTneuropathy score, the neuropathy was mild in 67% (n=30), moderate in 31%(n=14) and severe in one case. 42% of patients (n=19) needed ankle-footorthosis to sustain feet during walking, one patient required unilateralsupport and one patient was wheelchair dependent. Detailed nerveconduction studies were available in 42 patients and invariably showed amotor axonal neuropathy, with intermediate reduction of conductionvelocities in 26% (n=11) and decreased sensory action potentials in 26%(n=65).

Example 2: Assessing SORD Protein Expression in Human Fibroblasts andSORD Levels in Blood

Sorbitol dehydrogenase is a homotetrameric enzyme of 38-kDa subunits,which is widely distributed in mammalian tissues (Johansson et al. 2001;Hellgren et al. 2007; Lindstad, Teigen, and Skjeldal 2013). Itrepresents the second enzyme of the two-step polyol pathway, in whichglucose is converted to sorbitol, a relatively non-metabolizable sugar,by the enzyme aldose reductase (AR). Sorbitol is then oxidized tofructose by SORD (FIG. 2A). To gather further insights into thefunctional consequences of recessive mutations in SORD, next SORDexpression was assessed in fibroblasts from five unrelated affectedindividuals with homozygous c.757delG; p.(Ala253GlnfsTer27) (n=4) orc.757delG; p.(Ala253GlnfsTer27) & c.895C>T; p.(Arg299Ter) (n=1) variantsas well as two unaffected carriers of c.757delG; p.(Ala253GlnfsTer27) inheterozygous state. SORD protein was absent in all patients and thewild-type levels were reduced in unaffected carriers compared tocontrols (FIG. 2B). Accordingly, intracellular sorbitol concentrationswere over 10 times higher in patients' fibroblasts compared to controls,in keeping with a loss of SORD enzymatic activity (FIG. 2C). Fastingsorbitol levels in serum from ten patients carrying the homozygousp.Ala253GlnfsTer27 mutation and ten unrelated controls and found it wasover 100 times higher (14.82±0.780 vs 0.046±0.004 mg/L, p<0.0001) wasdetermined, confirming the lack of SORD enzymatic activity in patients(FIG. 13). This study also demonstrates that sorbitol is a useful markerfor detecting or characterizing inherited neuropathy associated withSORD mutation in a mammalian subject.

Example 3: Investigating the SORD Mutation in Models of SORD Deficiency

To further explore the pathophysiology of SORD mutation in vivo,Drosophila melanogaster models of SORD deficiency were established.Drosophila has two functional SORD genes (Sodh1 and Sodh2) that share90% residue identity (Luque et al. 1998). SORD is conserved acrossdistant phyla and Drosophila Sodh1 (NP 001287203.1) and Sodh2 (NCBIReference Sequence: NP 524311.1) encoded proteins share 75% and 73%identity with human SORD protein (NCBI Reference Sequence: NP 003095.2(SEQ ID NO: 46)), respectively. A mutant allele of Sodh2 was obtainedwhere the gene is disrupted by a transposon Minos mediated integrationcassette (MiMIC) insertion (Sodh2^(MB01265)) (Bellen et al. 2011).Homozygous Sodh2 (Sodh2^(MB01265/MB01265)) mutants are viable withnormal life span. To characterize neurodegenerative phenotypes, theDrosophila visual system was used to take advantage of the highlyorganized parallel axons of the compound eye that allow in vivodetection of subtle neuronal and synaptic pathological changes(Bausenwein, Dittrich, and Fischbach 1992). Axons of the outerphotoreceptor axons traverse the lamina cortex and make synapticconnections with lamina monopolar neurons in the lamina layer (FIG. 3A).In the control flies (yw) at 2 days after eclosion (DAE), the organizedlamina cartridges of photoreceptor synapses can be visualized in the xy-and xz-planes, respectively (FIG. 3B). A loss of photoreceptor terminalsin the lamina layer of Sodh2^(MB01265/MB01265) mutants was observed at 2days after eclosion (DAE) (FIG. 3C). The phenotype became progressivelysevere at 10 DAE, with vacuoles being more numerous and larger in sizedistributed across the synaptic lamina layer (FIG. 3C, D). Thesevacuoles exhibited a loss of neuronal membrane (marked by HRPlabelling), as well as a reduced Bruchpilot (BRP, a synaptic active zonecytomatrix protein) labelling, indicating synaptic degeneration (FIG.3C, D). To validate the findings described herein, a second SORD modelwas generated by specific knockdown of both Sodh1 and Sodh2 expressionin neurons using a pan-neuronal driver elav^(C155). Loss of both Sodh1and Sodh2 resulted in age-dependent synapse degeneration, similar tothat of homozygous Sodh2 (Sodh2^(MB01265/MB01265)) (FIG. 6). Thebehavioral phenotypes of SORD deficiency the Sodh2^(MB01265/MB01265)homozygous flies with a systemic loss of function in Sodh2 werecharacterized and although these flies exhibited a normal life-span,their locomotor activity was significantly compromised at a late-stage(40 DAE) (FIG. 3E, F). This indicated a progressive, age-dependentneuromuscular dysfunction reminiscent of hereditary neuropathies.Moreover, the sorbitol levels were measured in fly heads at 10 DAE andobserved a significant increase in the Sodh2^(MB01265/MB01265) model(FIG. 4B), consistent with the observation in patient fibroblasts. Takentogether, Drosophila models of SORD deficiency were successfullyestablished that recapitulate typical pathological phenotypes in humanpatients, including (1) a normal lifespan, (2) progressive andage-dependent synaptic degeneration and locomotor deficiency, and (3)increased sorbitol levels.

After establishing loss-of-function as the mechanism of action and aknown enzymatic pathway, treatment options for SORD associatedhereditary neuropathy were investigated. It had previously been shownthat the pharmacological inhibition of aldose reductase, the enzymeupstream of SORD, represents a successful strategy to reduce toxicsorbitol accumulation in cellular and animal model of diabetes (Kikkawaet al. 1983; Matsumoto et al. 2008; Ramirez and Borja 2008; Hao et al.2015; Grewal et al. 2016) and, arguably, also humans (Chalk, Benstead,and Moore 2007; Polydefkis et al. 2015; Sekiguchi et al. 2019). Theeffect of two commercially available aldose reductase inhibitors (ARI),Epalrestat and Ranirestat, were tested on intracellular sorbitolaccumulation in patient fibroblasts lacking functional SORD. Patient andcontrol fibroblasts were grown for 72 hrs in the presence or absence ofEpalrestat (100 μM) or Ranirestat (10 μM) and intracellular sorbitollevels were measured thereafter. Both ARI, Epalrestat and Ranirestat,achieved a significant reduction of sorbitol to a level comparable tocontrols (FIG. 4A). Further, Drosophila models of SORD were fed withEpalrestat and Ranirestat starting at 2 DAE. A significant reduction ofsorbitol level was observed in the Sodh2^(MB01265/MB01265) fly heads at10 DAE (FIG. 4B). Importantly, the locomotor activities ofSodh2^(MB01265/MB01265) flies and flies with neuronal specific knockdownof both Sodh1 and Sodh2 were rescued to the levels of yw control flies(FIG. 4C, FIG. 7). Furthermore, Epalrestat or Ranirestat feedingrestored the age-dependent synaptic defects in Sodh2^(MB01265/MB01265)mutant flies. In DMSO vehicle treated flies, the loss of synaptictermini was highly prominent in the advanced age of 40 DAE where theexpansion of neighboring vacuoles resulted in fused, much largervacuoles encompassing multiple synaptic cartridges (FIG. 4D).Remarkably, epalrestat/ranirestat feeding reduced the number of vacuolesand restored the localization of synaptic cytomatrix protein BRP at both10 and 40 DAE (FIG. 4E-G).

In summary, SORD represents a novel recessive gene causingaxonal/intermediate, motor predominant CMT. Genetic data from the cohortas well as from control databases suggest that the predominantpathogenic variant in SORD, c.757delG; p.(Ala253GlnfsTer27), with acarrier frequency in of ˜3/1,000 individuals in the population, mayrepresent one of the most common specific alleles causing a recessiveMendelian disease. Indeed, with a frequency in undiagnosed CMT2 and dHMNcases of up to ˜10%, it will likely account for a significant portion ofthe diagnostic gap in inherited axonal neuropathies. It is intriguingthat, despite their frequency, mutations in SORD were not identified asa cause of CMT by previous studies. The presence of the human SORD2Pgene duplication may have hampered the detection of variants in thefunctional SORD, since available annotation programs are highlydependent on the unique mapping of 150-300 bp long reads generated bycurrent next-generation sequencing technologies. Other known pathogenicvariants have previously been shown to be concealed by the presence ofpseudogenes (De Vos et al. 2004). The pathogenicity of SORD mutations isfurther supported by in vitro data in patient-derived fibroblasts, whichshowed absent SORD protein and intracellular sorbitol accumulation. Twoin vivo Drosophila models recapitulated the human phenotype withprogressive synaptic degeneration and motor impairment, SORD deficiency,and in increased sorbitol levels.

The studies described herein demonstrate that enzymatic loss-of-functionand subsequent sorbitol accumulation is a mechanism of action for SORDassociated CMT. Previous studies in cellular and animal models ofdiabetes have shown that an increased polyol influx with intracellularsorbitol accumulation is paralleled by an increase in cellularosmolarity, oxidative stress and decreased NADPH levels, which can allhave a detrimental effects on peripheral nerves (Schmidt et al. 2001;Obrosova 2005; Sango et al. 2006). However, previous studies on adultC57BL/LiA mice expressing reduced level of SORD protein due to anintronic splicing mutation did not identify overt neurological defects(Holmes, Duley, and Hilgers 1982; Lee, Chung, and Chung 1995; Ng et al.1998). Based on patient clinical data and the late-onset phenotype inflies, it will be important to extend the observation to aging C57BL/LiAmice or create complete knock-out SORD mouse or rat models. The studyfurther unravels a central role of the polyol pathway in peripheralnerve metabolism and survival in normoglycemic conditions. Although themechanism by which intracellular sorbitol accumulation can lead toselective degeneration of peripheral nerves is yet unknown, theobservation of increased sorbitol levels in patient derived cells inthis study has promising implications, both as a biomarker of thedisease and as a target of future therapeutic interventions, includingmethods for substrate reduction, gene replacement or correction, andSORD enzyme substitution. Accordingly, disclosed herein are preclinicalstudies demonstrating the beneficial effects of substrate reduction viaARI application in human derived cells and Drosophila models. Epalrestatis currently marketed in few countries for the treatment of diabeticcomplications (Grewal et al. 2016) while Ranirestat has been advancedinto late stages of clinical trials (Polydefkis et al. 2015; Sekiguchiet al. 2019).

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1. A method of treating inherited neuropathy in a mammalian subject, themethod comprising: (a) detecting the presence of a mutation in thesorbitol dehydrogenase (SORD) gene in a sample from the subject; and (b)administering to the subject a polynucleotide that encodes a SORDpeptide, an aldose reductase antisense oligonucleotide, an agent thatblocks expression of a mutant SORD gene, an agent that corrects themutation in SORD gene, or a combination of any of the foregoing.
 2. Themethod of claim 1, wherein the method comprises administering apolynucleotide encoding the SORD peptide.
 3. The method of claim 1,wherein the method comprises administering an agent that corrects themutation in the SORD gene, wherein the agent is a CRISPR Cas9 proteinand one or more guide RNA molecules.
 4. A method of treating inheritedneuropathy in a mammalian subject, the method comprising administeringto the subject an aldose reductase inhibitor.
 5. A method of treatinginherited neuropathy in a mammalian subject, the method comprising: (a)detecting the presence of a mutation in the sorbitol dehydrogenase(SORD) gene in a sample from the subject; and (b) administering to thesubject a SORD peptide.
 6. The method of claim 1, wherein the mutationin the SORD gene is c.753delG; p.Ala253GlnfsTer27, c.329G>C;p.Arg110Pro, c.298C>T; p.Arg100Ter, or c.458C>A; p.Ala153Asp.
 7. Themethod of claim 1, wherein the mutation in the SORD gene is c.757delG;p.Ala253GlnfsTer27, c.28C>T; p.Leu10Phe, c.316_425+165del; p.Cys106Ter,c.295C>T; p.Arg299Ter, c.964G>A; p.Val322Ile, or a deletion ofindividual or multiple coding exons or the entire SORD gene.
 8. Themethod of claim 1, further comprising measuring sorbitol in a samplefrom the subject.
 9. A method of characterizing a neuropathy in amammalian subject, the method comprising measuring the level of sorbitolin a subject suffering from a neuropathy, wherein a sorbitol level ofgreater than about 10 g/L indicates that the neuropathy is associatedwith a mutation in the sorbitol dehydrogenase (SORD) gene.
 10. A methodof evaluating the efficacy of a treatment for an inherited neuropathy ina subject, the method comprising administering to the subject an agentselected from the group consisting of an aldose reductase inhibitor, analdose reductase antisense oligonucleotide, a polynucleotide thatencodes a SORD peptide, a SORD peptide, an agent that blocks expressionof a mutant SORD gene, and an agent that corrects the mutation in SORDgene, or a combination of any of the foregoing; and measuring the levelof sorbitol in a subject.
 11. The method of claim 4, comprisingdetecting the presence of a mutation in the sorbitol dehydrogenase(SORD) gene in a sample from the subject.
 12. The method of claim 4,wherein the aldose reductase inhibitor is selected from the groupconsisting of alrestatin, epalrestat, diepalrestat, fidarestat,imirestat, lidorestat, minalrestat, ponalrestat, ranirestat, salfredinB₁₁, sorbinil, tolrestat, zenarestat, and zopolrestat.
 13. The method ofclaim 5, wherein the mutation in the SORD gene is c.753delG;p.Ala253GlnfsTer27, c.329G>C; p.Arg110Pro, c.298C>T; p.Arg100Ter, orc.458C>A; p.Ala153Asp.
 14. The method of claim 5, wherein the mutationin the SORD gene is c.757delG; p.Ala253GlnfsTer27, c.28C>T; p.Leu10Phe,c.316_425+165del; p.Cys106Ter, c.295C>T; p.Arg299Ter, c.964G>A;p.Val322Ile, or a deletion of individual or multiple coding exons or theentire SORD gene.
 15. The method of claim 5, further comprisingmeasuring sorbitol in a sample from the subject.
 16. An adeno-associatedviral (AAV) vector that encodes a SORD peptide.
 17. An isolated nucleicacid comprising an antisense oligonucleotide sequence listed in Table 2or 16-18.